Stabilization of animal and poultry feeds containing antibiotics



United States Patent M 3,157,512 STABEIZATION OF ANIMAL AND POULTRY FEEDS CONTAJNING ANTlBlGTlQS Siegfried Arthur Muller, Cluster, N.Y., amignor to American Cyanamid Company, Stamford, Conrn, a corporation of Maine No Drawing. Griginal application June 22, 1960, Ser. No. 37,853. Divided and this application Dec. 1, 1961, Ser. No. 157,868

' 1i) (Ilaims. ((Il. 99-9) This invention relates to the stabilization of animal and poultry feed supplements and to animal and poultry feeds containing a tetracycline type antibiotic and more particularly is concerned with a novel process of prepar- {ng animal feed supplements and/or animal feeds which are stable upon prolonged storage against loss of antibiotic potency.

In recent years the use of antibiotics in animal feeds for improving growth characteristics and efficiency of feed utilization has become of considerable economic importance. One of the best of such procedures is described-inthe United States patent to Jukes, No. 2,619,- 420, which involves the addition to. animal feeds of the antibiotics produced by fermentation of an aqueous nutrient medium with the microorganism Streptomyces aureofaciens. Inpractice, the fermentation mash solids are harvested, after the fermentation has been continued for-the requisite length of time, and dried. ,The dried harvest mash solids containing the antibiotics produced by the microorganism-Streptomyces aureofaciens can be used as such an animal feed supplement or they may be blended with conventional animal feed materials to produce a nutritionally-balanced growth accelerating animal feed.

The use of the dried chlortetracycline-containing fermentation harvest mash solids as an animal or poultry feed supplement has from the beginning presented very vexing problems particularly the loss of potency of the antibiotic in the feed orfeed supplement upon prolonged storage; This loss of antibiotic potency, and which frequently runs as high as 20% over a one-year storage period, has heretofore necessitated the addition of a suitable overage of-dried harvest mash antibiotic-containing solids to the animal feed blend so as to maintain the antibiotic potency'during storage. Such aprocedure is unsatisfactory and among other things results in higher than desired'manufacturing costs for these animal feed supplements.

In commercial practice, a typical procedure for obtaining the dried harvestmash-antibiotic-containingsolids is carriedout by harvesting the fermentation mash at the prevailing pH, that is about pH 68. A filter aid such as diatomaceous earthis then'added and'the solids are collected by filtration and dried. The dried filter cake containing from l grams of chlortetracycline per pound may be marketed as such or may be blended with conventional poultry or animal feed materials to produce a nutritionally-balanced growth accelerating antibiotic containing animal feed. H H v I p The presentinventionis based upon the surprising discovery that instead of harvesting and drying the fermentation mash at the Weakly alkaline pH of about 6-8, if the mash is first acidified to a pH of about 0.1-3 and then readjusted upward to an alkaline pH of around 8.5-

3,l57,5l2 Patented Nov. 17, 1964 13, the dried mash solids are stabilized against loss of antibiotic potency upon storage for prolonged periods of time. It is not known with any degree of certainty the reason why the novel down-up process of this invention results in the stabilization of the harvest mash solids to such a considerable extent but it is believed that it involves the formation of a calcium-antibiotic-carrier complex of some sort which requires acid conditions. In any event, the resulting dried mash solids are remarkably stable towards loss in antibiotic potencies'.

it has been determined that the optimum acid pH is about 2 or lower and the necessary or minimum upper pH is around 8.5 or slightly higher. Specific hydrogen ion concentrations vary slightly with different tetracyclines. .For example, with chlortetracycline a pH of has been found to result in enhanced stability. For tetracycline a pH of 9.0 has been found to be best while with oxytetracycline a pH of 9.5 produces optimum results. With demethylchlortetracycline a pH of 8.7 has proven satisfactory.

The particular acid employed to lower the pH of the fermentation harvest mash to the desired level is a factor of some importance. While, in general, any mineral acid may be used, best results are obtained when hydrochloric acid or sulfuric acid is used. This may be because certain acids such as phosphoric acid have a tendency to remove calcium from the mash and which seems to be atleast in part responsible for the desirable results obtained by the process of this invention.

In carrying out the present invention the fermentation harvest mash, and which at harvest usually has a pH varying from about 6.5 to 7.5, is first adjusted downward to a pH of around 2 or lower with hydrochloric acid or sulfuric acid. A short time later, for example, 2-5 minutes, with stirring, the mash is adjusted upward with caustic such as an alkali metal hydroxide or alkaline earth metal hydroxide to a pH of around -13 depending upon the particular antibiotic contained in the mash. The alkaline mash is then slurried with 24% (tin/v.) of diatomaceous earth and if desired with 0.2% of a synthetic hydrated magnesium silicate such as magnesol which has the approximate composition The slurry is then filtered and the filter cake dried at a temperature ranging from about 25 to about C. and the moisture content is adjusted to a range of from 5% to 20%.

It has been found that when samples of the dried harvest mash solids obtained by the novel process of this invention are assayed for antibiotic potency after storage at 56 C. for one week, the average loss in potency is only 6% whereas standard production material and obtained by the commercial process hereinbeforedescribed shows an average loss of antibiotic potency of 24%.

While the process of this invention results in a considerable improvement in stability of the feed supplement, that is, the dried harvest mash solids containing from 1-50 grams of antibiotic per pound, and which makes it possible for the firsttime to storethese feed supplements for extended periods of time without appreciable loss in antibiotic potency, nevertheless, when the feed supplement is blended with conventional poultry feed materials it has been found that there is a distinct loss in antibiotic potency of the finished feed upon storage for prolonged periods of time.

lthas been discovered that this loss in antibiotic potency of the finished feed can be prevented by the addition of mild caustic, preferably calcium hydroxide, to the finished feed in a quantity sufficient to raise and maintain the pH to between 6 and 12 and preferably 8.5-1l.5. The combination treatment of this invention results in the stabilization of the feed supplement so that it is stable upon storage and when this treated feed supplement is blended with conventional poultry feed materials the finished feed can likewise be stabilized by the described treatment with. calcium hydroxide. a

The invention will be described in greater detail in conjunction with the following specific examples.

7 Example 1 A l-liter portion of harvest mash obtained as a result of Streptomyces aureofaciens strain A-377 fermentation for 120 hours at 28 C. was adjusted to pH 7.2 with 15 N sodium hydroxide solution. A 20-grain quantity of diatomac'eous earth filter-aid and, a Z-gram quantity of mag nesium silicate were added. The slurry was stirred for 10 minutes, then filtered. The filter cake was air-dried overnight to a volatiles content. of 8.5 This product assayed 92 mcg. of chlortetracycline per milligram of solids by microbiological assay. After standing in a tightly-capped jar at 56 C. for 7 days, the product assayed 65 mcg./mg., a loss of 26% of the original activity.

Another l-liter portion of this harvest mash was adjusted to pH l.7 by the addition of concentrated hydrochloric acid. AZO-gram quantity of diatomaceous earth filter-aid and 2 grams of magnesium silicate were added and the s'lurry'stirred. After 5 minutes of stirring, 15 N sodium hydroxide solution was used to adjust the pH to 8.4. The slurry was filtered and the filter cake dried to a volatiles content of 7.8%. This cake assayed 90.5 mcg. of chlortetracycline per milligram of solids. After being kept 7 days in a tightly-capped jar at 56C.,this product assayed 89 mcg./mg.; a loss of only 1.7% of the original activity as compared to the 26% loss in the control.

Example 2 A l-Iiter portion of chlor-tetracycline-containing 120- 7 hour at 28 C. harvest mash was adjusted to pH 7.3 with to pH 1.4 with sulfuric acid, stirred five minutes, and then adjusted to pH 8.4 with 15 N sodium hydroxidesolution.

The same quantity of diatomaceous earth was added as before. Filtration and drying produced a cake having 8.4% volatile matter. This cake assayed 731mcg./rng. before standing at 56 C. for 7 days and 71.5 meg/mg.

. afterwards, for a loss of 2.1% of the original activity as compared tothe 23% loss in the control.

I Example 3 7 Three l-liter portions of a 120-hour (28 C.) tetracycline-containing fermentation harvest mash were treated as follows:

(a) A l-liter portion was adjusted to pH 7.6 with so- 7 dium-hydroxide solution, slurried with 40 grams of diatomaceous earth filter-aid and 2 grains of magnesium" silicate. stirred, and filtered. The filter cake was air-dried V and adjusted to a moisture content of 8.5%.

silicate, stirred, and filtered. The filter cake was airdried and adjusted to a moisture content of 8.5

(c) A third l-liter portion-was adjusted to pH 1.5 with hydrochloric acid, then adjusted to pH 9.0 with sodium hydroxide. solution, slurried with 40 grams of diatornac eous earth filter aid and 2 grams of magnesium silicate, stirred and filtered. The filter cake wasair-driedand adjusted to a moisture content of 8.5 V

All three filter cakes were assayed (microbiologi'cally) for tetracycline content, allowed to stand for 7 daysat 56 C. intightly-capped jars, then assayed again (microbiologically) to determine potency losses, with the following results.

T m ass ssinate (a) pH 7.6 26 (b) pH 10.0 2 (c) pH 2.0 to pH 9.0 7

Example 4 i j V Three l-liter portions of e hour (28 C.) oxytetracycliue-containing fermentation harvest mash were treated as follows: V j (a) A l-liter portion was adjusted to pH 7.6 with soatomaceous earth and 2 grams of magnesium silicate; stirred, and filtered. The filter cake was air-dried and adjusted to a moisture content of 8.5% V (b) A second l-liter portion was adjusted to pH 9.5 with sodium hydroxide solution, surried with 40 grams of diatomaceous earth and 2 grams of magnesium silicate,

' stirred and filtered. The filter cake was air-dried and ad.-

Loss in potency in 7 days Treatment: I at 56 C., percent' (a) pH 7.6 33 (b) pH 9.5 .55 v (C) pH 2.0-pH 9.5 L 9 Example 5 A A Two l-liter portionsof a 120-hour (28 C.) demethyl chlortetracycline containing fermentation harvest mash were treated as follows:

(a) A l-liter portion was adjusted to pH 7.6 with sodium hydroxide solution, slurried with 30 grams of diatomaceous earth, and filtered. The filter cake'was dried I and its moisture content then adjusted to 8.5

(b) A second l-liter portion was adjusted to pH 1.9 with hydrochloric acid solution then, within 5 minutes, adjusted to pH 8.7 with sodium hydroxide solution and" slurried with'30 grams of diatomaceous earth, The slurry was filtered and the filter cakethus obtained was dried as above. Subsequently, the moisture content of the dried filter cake'wasadjusted to 8.5

7. Both filter cakes were assayed for demethylchlortetracycline content, allowedto stand for 7 days-at 56- in 1 tightly-capped jars, then assayed to determine potency.

losses. Test results were as follows.

Loss in potencyin 7 days Treatment: C. percent a (a) pH 7.6 .'15

' j (b) pH 1.9 rr 8.7 6

Example]? 7 QFeed supplements were, prepared as in Example 1 j 7 using both the conventionalprocedure and (the process of V V dium hydroxide solution, slurried with 40 grams of di- K Initial Loss in Assay for Potency at OTC, 56 C. for mcgJg. 7 days, percent Feed Supplement Type Treatment Conventional New Process Then chick feeds were prepared; using the conventional procedure solids and new process solids as ingredients; in the proportions of 100 grams of standard chick feed and 0.5 gram of solide. The standard chick feed consisted of 65% yellow corn, 20% soybean oil meal, corn gluten meal, 5% fish meal, 2% alfalfa meal, 1.5% steamed bone meal, 2% calcite, and a miscellaneous group of essential materials such as minerals, vitamins, and the like. These two types of feeds were assayed for chlortetracycline content then kept at moisture and 56 C. for 7 days in tightly-closed containers and again assayed with the following results.

Loss in7potency at 56 C. for

Feed intermediate type: days, percent Conventional New Process 21 and thus maintaining, by means of the Ca(OH) a pH of 9.0 in the final chick feed blends. Both blends were kept at 10% moisture and 56 C. for 7 days in tightlyclosed containers; with the following results.

Loss in potency at 56 C. for Feed yP 7 days, percent Conventional 26 New process 2 Example 7 Feed supplements were prepared as in Example 1, using both the conventional procedure and the process of the present invention to obtain chlortetracycline-containing harvest mash solids having moisture contents of about 17%.

Then animal feeds were prepared, using the conventional procedure solids and new process solids as ingredients, in 1:1 proportions with soybean feed containing 1.5% lard oil. The moisture content of both the animal feed blend containing conventional procedure solids and the animal feed blend containing the new process solids was 10%. These two feed blends were assayed for chlontetracycline content, held at 56 C. for 7 days in tightly-closed containers, and again assayed. Test results were as follows.

Feed typ Conventional 13 New Process 3 Example 8 The addition of 5% dry calcium hydroxide to harvest mash solids (feed supplements prepared in accordance with the present invention) was carried out in a number of runs. The results obtained are shown 'in the table below.

Percent Percent Recovery Recovery 49 Days, 49 Days,

(Control) It will be seen that there is an average of a 25% loss in potency in the control and only a 10% loss in the samples treated with calcium hydroxide.

I claim:

1. A process for the production of dried fermentation harvest mash solids containing a tetracycline antibiotic which comprises adjsting the pH of the harvest mash to a pH of around 0.1-3 with a mineral acid raising the pH of the acidulated harvest mash to a pH of around -13 and thereafter filtering and drying the harvest mash so as to stabilize the mash solids against loss in antibiotic potency.

2. A process for the production of dried ermentation harvest mesh solids containing a tetracycline antibiotic 'which comprises adjusting the pH of the harvest mash to a pH of around 0.1-3 with an acid of the group consisting of hydrochloric acid and sulfuric acid, raising the pH of the acidulated harvest mash to a pH of around 85-13 with caustic and thereafter filtering and drying the harvest mash so as to stabilize the mash solids against loss in antibiotic potency.

3. A process according to claim 2 in which the tetracycline antibiotic is chlortetracycline.

4. A process according to claim 2 in which the tetracycline antibiotic is tetracycline.

5. A process according to claim 2 in which the tetracycline antibiotic is oxytetracycline.

6. A process according to claim 2 in which the tetracycline antibiotic is demethylchlortetracycline.

7. A process according to claim 2 in which the tetracycline antibiotic is demethyltetracycline.

8. A process for the production of an animal feed composition which comprises adjusting :the pH of a fermentation harvest mash containing the growth-promoting factors produced by elaboration of a microorganism of the group consisting of Streptomy ces aureofaciens and Streptomyces rimosus to a pH of around 0.1-3, raising the pH of the acidulated harvest mash to a pH of around 85-13, thereafter filtering and drying the harvest mash solids to produce an animal feed supplement, adding to said animal feed supplement a major amount of an edible animal feedstuff and sufiicient mild caustic so as to maintain the pH of the animal feed between pH 6-12 whereby 7 2 the animal feed is stabilized against losses in antibiotic potency for extended periods of time.

9. A process according to claim 8 in which the caustic "is calcium hydroxide.

10.,A process for the production of an animal feed composition which comprises adjusting the pH of a fermentation harvest mash containing chlortetracycline to apH of around 0.1-3, raising the pH of the aciduiated hydroxide so as to maintainthe pH between pH 6-12 whereby the animal feed is stabilized against losses in antibiotic potency for extended periods of time. 2

References Cited in the file of this patent UNITED STATES PATENTS I Iukcs Nov. 25, '1952' Hntanen et a1. Nov. 29, 1960 Chertow Feb. 7, 1961 McCormick Feb. 7, 1961 Klothen Jan. 30, 1962 

1. A PROCESS FOR THE PRODUCTION OF DRIED FERMENTATION HARVEST MASH SOLIDS COTIANING A TETRACYCLINE ANTIBIOTIC WHICH COMPRISES ADJUSTING THE PH OF THE HARVEST MASH TO A PH OF AROUND 0.1-3 WITH A MINERAL ACID RAISING THE PH OF THE ACIDULATED HARVEST MASH TO A PH OF AROUND 8.5-13 AND THEREAFTER FILTERING AND DRYING THE HARVEST MASH SO AS TO STABILIZE THE MASH SOLIDS AGAINST LOSS IN ANTIBIOTIC POTENCY. 